The present invention relates to pharmaceutical compositions comprising synthetic peptides derived from vitronectin capable of modulating the biological activities of plasminogen activator inhibitor-1 (PAI-1) and to some such novel synthetic peptides.
Hemostasis, the process whereby bleeding from an injured blood vessel is arrested, is characterized by the combined activity of vascular, platelet and plasma factors as well as counterbalancing mechanisms to limit the accumulation of platelets and fibrin to the area of vessel wall injury.
Blood coagulation reactions form a key element of the hemostatic seal-the fibrin clot. Spreading outward from and anchoring the platelet plugs, the fibrin clot adds bulk needed for the hemostatic seal.
Regulatory mechanisms normally exist to prevent the blood coagulation reactions, once activated, from proceeding unchecked to cause pathological conditions, such as local thrombosis or disseminated intravascular coagulation (DIC). These regulatory mechanisms include cellular clearance of activated clotting factors, particularly as blood flows through the liver, and mechanisms within the blood itself for neutralizing the activated enzymes and cofactors of blood coagulation.
The fibrinolytic system is activated following the deposition of fibrin. By dissolving fibrin, this system helps keep open the lumen of an injured blood vessel. A balanced deposition and lysis of fibrin maintains and remolds the hemostatic seal during the several days required for an injured vessel wall to be repaired.
When fibrinogen is converted to fibrin by thrombin, plasminogen, an inert plasma precursor of plasmin, is activated by its natural activators, e.g., tissue plasminogen activator (tPA), and converted to the powerful proteolytic enzyme, plasmin, which degrades fibrin into soluble fragments, referred to as fibrin degradation products, which are swept into the circulation.
Several factors operate to prevent excessive fibrinolysis, including increased affinity of plasminogen for binding to fibrin rather than to fibrinogen, and the increased ability of tissue plasminogen activator to activate plasminogen when it is bound to fibrin. A major inhibitor of fibrinolysis is plasminogen activator inhibitor-1 (PAI-1). Moreover, plasma contains a protease inhibitor called .alpha.2-antiplasmin that can rapidly inactivate plasmin escaping from a fibrin clot.
In order to stop plasmin action it is not sufficient only to inactivate the excess of already-formed plasmin by .alpha..sub.2 -antiplasmin (Collen, 1976). It is also necessary to arrest further production of plasmin from plasminogen. This is achieved through the inhibition of plasminogen activator(s) by PAI-1, which was shown to be bound to vitronectin (Loskutoff et al., 1988; Sprengers and Kluft, 1987) and thus to become stabilized in its active form both in circulating blood and in the extracellular matrix (ECM) (Declerck et al., 1988; Salonen et al., 1989; Mimuro and Loskutoff, 1989; Preissner et al., 1990).
Originally discovered as a cell spreading factor, vitronectin is now recognized as a multifunctional regulatory protein involved in a variety of extracellular processes such as the attachment and spreading of normal and neoplastic cells, as well as the function of the complement and coagulation pathways. In circulating blood, vitronectin occurs in two molecular forms: a single chain 75 kDa polypeptide (V.sub.75) and a nicked polypeptide (V.sub.65+10) in which the two chains (65 kDa and 10 kDa) are linked by an interchain disulfide bridge. Using the specific phosphorylation of vitronectin at Ser.sup.378 by platelet-released protein kinase A (PKA) (Korc-Grodzicki et al., 1988a; Korc-Grodzicki et al., 1988b; Chain et al., 1990; Korc-Grodzicki et al., 1990; Chain et al., 1991a), we recently showed (Chain et al., 1991b) that plasmin specifically cleaves vitronectin at the Arg.sup.361 -Ser.sup.362 bond, 18 amino acids upstream from the site of the endogenous cleavage which gives rise to the two-chain form (V.sub.65+10) of this protein. We also reported that as a result of the plasmin cleavage, the affinity between vitronectin and PAI-1 is significantly reduced (Chain et al., 1991b) and that this cleavage is stimulated by glycosaminoglycans which anchor vitronectin to the ECM, thus favouring the cleavage of the vitronectin molecules found in this matrix. On the basis of these findings, we proposed the mechanism depicted in FIG. 1, through which plasmin can arrest its own production by feedback signalling.
At the initial stage of fibrinolysis, the plasminogen activator converts plasminogen to plasmin. This is made possible since PAI-1 is then anchored (trapped) by the vitronectin molecules which were previously shown to be immobilized in the ECM (Pollanen et al., 1988), presumably through glycosaminoglycans. This anchoring of PAI-1 locally depletes the inhibitor by preventing it from reaching and inhibiting the plasminogen activator. When plasmin levels become too high, the excess plasmin can clip preferentially the vitronectin molecules immobilized in the subendothelium. Consequently, the equilibrium between anchored PAI-1 and the detached (mobile) PAI-1 is displaced, thus unleashing PAI-1 to be transferred to soluble (mobile) vitronectin molecules in plasma which can then reach and inhibit the plasminogen activator and arrest plasmin production. This unleashing actually represents a translocation, a transfer of the anchored PAI-1 from the ECM-bound (plasmin-clipped) vitronectin to the soluble vitronectin molecules which have not been clipped by plasmin and thus possess a higher affinity for PAI-1.
The impairment of this control mechanism of plasmin production may have wider clinical implications. Several findings suggest that the activation of plasminogen by its natural activators (e.g., tPA, streptokinase, urokinase) constitutes a multipurpose biological tool for the proteolytic dissolution of barriers by plasmin. In the case of blood clots, whether accidentally formed or no longer needed, their dissolution restores the free and vital flow of blood. However, the very same mechanisms is apparently exploited by malignant cells for penetrating tissues and forming metastases (Dano et al., 1985; Mignatti et al., 1989; Cajot et al., 1990), by nerve cells in nerve regeneration, and by blood vessels in angiogenesis (Mignatti et al., 1989). The plasminogen/plasmin system was also shown to be involved in inflammation, ovulation, tissue remodeling and development.
In view of this diversity of functions, it is obvious that plasmin activity must be under a strict regulatory control to secure a localized, site-restricted action and to limit its duration.
PAI-1 has an important role in the regulation of in plasminogen activation and as such, it is involved regulating the dissolution of biological barriers by plasmin. Consequently, through its functional interaction with PAI-1, vitronectin is involved in fibrinolysis, inflammation, ovulation, tissue remodeling and development, angiogenesis, nerve regeneration, malignancy and tumor cell invasion (Dano et al, 1985, Mignatti et al, 1989, Cajot et al, 1990).
Some peptides derived from the heparin binding site of vitronectin have been recently shown to mediate binding of vitronectin-thrombin-antithrombin III complex to human endothelial cells (de Boer et al, 1992). The peptides disclosed by de Boer correspond to sequences A.sup.341 -R.sup.355 (1-15 of SEQ ID NO:1), K.sup.348 -R.sup.361 (8-21 of SEQ ID NO:1, R.sup.357 -R.sup.370 (17-30 of SEQ ID NO:1), H.sup.366 -R.sup.379 (26-39 of SEQ ID NO:1), and N.sup.371 -L.sup.383 (31-43 of SEQ ID NO:1). These same peptides were also used in a mapping study to localize the binding sites for heparin, PAI-1 and plasminogen in the carboxyl-terminal portion of vitronectin (Kost et al, 1992). None of these publications describe or suggest a pharmaceutical utility for these peptides.